CN110484521A - The phytic acid enzyme mutant KsPHY9 and its gene of high thermal stability and application - Google Patents
The phytic acid enzyme mutant KsPHY9 and its gene of high thermal stability and application Download PDFInfo
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Abstract
The present invention relates to field of biotechnology, and in particular to the phytic acid enzyme mutant KsPHY9 and its gene of high thermal stability and application.The amino acid sequence of the phytic acid enzyme mutant KsPHY9 is that amino acid sequence phytase KsPHY as shown in SEQ ID NO:1 includes following mutational site: A73P, A80P, D107K, N203D, G211S, Q224E, Q252V, T326Y, K379P.Compared with the 23.7% residual enzyme activity of parent's phytase KsPHY, the remnant enzyme activity of the phytic acid enzyme mutant KsPHY9 is 56.9%, and heat resistance greatly improves.
Description
Technical field
The present invention relates to field of biotechnology, and in particular to the phytic acid enzyme mutant KsPHY9 and its gene of high thermal stability
And application.
Background technique
Phytic acid (Phytate, Phytic acid, IP6) is also known as phytic acid, contains 6 phosphate groups, with rich
Rich phosphorus, is the important storage form of phosphorus in feed.Phytic acid is the major storage form of phosphorus in the crop seeds such as beans and cereal,
But nonruminant (such as poultry, pig) decomposes the enzyme of phytic acid due to lacking in vivo, so being difficult to efficiently use phytate phosphorus, big portion
Phosphorus existing for dividing in the form of phytic acid is expelled directly out in vitro by animal, causes serious environmental pollution.In addition, phytate phosphorus or one kind
Anti-nutritional factors, it can be with various metals ion (such as Ca2+、Mn2+、Mg2+、Zn2+、Cu2+、Fe2+Deng) and much protein
Corresponding insoluble complex is chelated into, the bioavailability for reducing these nutriments and animal are to nutriment
Effective rate of utilization.
Phytase (phytase), i.e. phytic acid hydrolase (myo-inositol hexakisphosphate
Phosphohydrolase, EC 3.1.3.8), it is that catalysis phytic acid (phytic acid) and its phytate hydrolysis generate inositol and phosphorus
The general name of the class of enzymes of sour (or phosphate), belongs to histidine phosphatase family.Phytase is added in feed, can not only be mentioned
High animal reduces pollution of the phosphorus to environment, can also reduce the anti-oxidant action of phytate phosphorus to the utilization rate of phytate phosphorus in feed.
Phytase animal feed, human health and in terms of show huge application prospect and researching value.
Most of phytase preparation being commercialized currently on the market is improved by gene optimization.Phytase is in feed
It need to usually be carried out at relatively high temperatures in granulating process, the denaturation of the phytase easy in inactivation of thermal stability difference, therefore filter out and have
The phytase of high thermal stability has great importance in scientific research and industrial application.
The zymoprotein of existing industrialization can be transformed using Protocols in Molecular Biology, acquisition can adapt to different productions
The enzyme of industry demand.Design and rational is the three-D space structure and the mechanism of action by studying zymoprotein, and enzyme activity may be influenced by finding out
Property or enzyme characteristic key amino acid, and to these amino acid carry out rite-directed mutagenesis, to obtain more preferably zymoprotein.With unreasonably
Property design compare, design and rational is more efficient, the result that often also can be more got well.
But be mutated compared to random gene, design and rational is to improve the success rate of experiment, and sequence analysis software cannot be protected
Demonstrate,prove the reliability of mutation.Theoretical prediction merely provides some possibilities, these prediction do not ensure that it is absolutely accurate, especially
When the case where mutation is more complicated, also Detailed Experimental is needed to verify the research of its property and function.
Summary of the invention
The object of the present invention is to provide the phytase KsPHY9 of high thermal stability.The high thermal stability phytase
KsPHY9 is to carry out rite-directed mutagenesis, the phytic acid enzyme mutant of acquisition to parent's phytase KsPHY.
The encoding gene of the object of the present invention is to provide a kind of phytic acid enzyme mutant KsPHY9 with high-fire resistance.
Another object of the present invention is to provide the weight of the phytic acid enzyme mutant KsPHY9 gene comprising above-mentioned heat-resisting raising
Group carrier.
Another object of the present invention is to provide the recombination of the phytic acid enzyme mutant KsPHY9 gene comprising above-mentioned heat-resisting raising
Bacterial strain.
Another object of the present invention is to provide a kind of method for obtaining phytic acid enzyme mutant KsPHY9 for expressing high heat resistance.
The present invention carries out molecule to phytase KsPHY shown in SEQ ID NO.1 using the method for fixed point saturation mutation and changes
It makes, obtains the phytic acid enzyme mutant KsPHY9 of heat-resisting raising by high flux screening, the phytase of heat-resisting raising of the invention is prominent
Variant KsPHY9 is compared with original Kosakonia sacchari phytase KsPHY, there is the difference of 9 amino acid, mutation
Amino acid sites include A73P, A80P, D107K, N203D, G211S, Q224E, Q252V, T326Y, K379P, the ammonia after mutation
Base acid sequence is as shown in SEQ ID NO.2.
The present invention also provides the recombinant vectors of the phytic acid enzyme mutant KsPHY9 comprising above-mentioned heat-resisting raising, will be of the invention
Heat-resisting raising phytic acid enzyme mutant KsPHY9 be connected to EcoR I and the Not I on Yeast expression carrier pPICz α A limitation
Between property restriction enzyme site, so that the nucleotide sequence is located at the downstream of AOX1 promoter and is regulated and controled by it, obtain expression of recombinant yeast
Plasmid pPICz α A-KsPHY9.
The present invention also provides the recombinant bacterial strains of the phytic acid enzyme mutant KsPHY9 comprising above-mentioned heat-resisting raising.According to this hair
Bright specific embodiment, the expression vector for expressing the phytic acid enzyme mutant are pPICZ α A；For the expression vector
The host cell of conversion is Pichia pastoris X33 and GS115.
The present invention also provides the methods for the phytic acid enzyme mutant KsPHY9 for expressing above-mentioned heat-resisting raising, including following step
It is rapid:
1) host cell is converted with above-mentioned recombinant vector, obtains recombinant bacterial strain；
2) it ferments to recombinant bacterial strain, induces the expression of recombinant phytase；
3) it after fermentation, recycles and purifies expressed phytic acid enzyme mutant KsPHY9.
The present invention carries out molecular modification, knot to Kosakonia sacchari phytase KsPHY by fixed point saturation mutation
Close the phytic acid enzyme mutant KsPHY9 that High Throughput Screening Assay filters out heat resistance raising.With parent's phytase KsPHY's
23.7% residual enzyme activity is compared, and the remnant enzyme activity of the phytic acid enzyme mutant KsPHY9 is 56.9%, and heat resistance greatly improves.
Detailed description of the invention
Fig. 1 is the Pichia pastoris X33 recombinant bacterium 7L tank fermentograph of the A-KsPHY9 of α containing pPICz.
Fig. 2 shows KsPHY9 enzyme solution and 80 water-bath enzyme activity retention rate of parent KsPHY enzyme solution.
Fig. 3 shows original phytase and the optimal reaction pH for being mutated phytase.
Fig. 4 shows that original phytase and the degrees of mutation phytase compare
Specific embodiment
The phytase that the method and thermal stability obtained that will be described present invention transformation phytase below improve.
Do not make the experimental methods of molecular biology illustrated in following embodiment, referring to " Molecular Cloning:A Laboratory guide "
Listed specific method carries out in one book of (third edition) J. Pehanorm Brooker, or carries out according to kit and product description；
The reagent and biomaterial commercially obtain unless otherwise specified.
Experimental material and reagent:
1, bacterial strain and carrier
Escherichia coli Topl0, OrigamiB, Pichia pastoris X33, carrier pPICZ α A, Zeocin are public purchased from Invitrogen
Department.
2, enzyme and reagent
Super 2 × Master of fidelity Mix PCR polymerase, restriction enzyme are purchased from NEB (Beijing) Co., Ltd；
Universai DNA Purification Kit, TIANprep Mini Plasmid Kit are purchased from biochemical (Beijing) section of Tiangeng
Skill Co., Ltd, other reagents are that domestic analysis is pure.
3, culture medium
Escherichia coli culture medium is LB culture medium (1% peptone, 0.5% yeast extract, 1%NaCl, pH7.0).LB+
Amp culture medium is the ampicillin that final concentration of 100ug/mL is added in LB culture medium.LB+Zeo culture medium is that LB culture medium adds
Enter the Zeocin of final concentration of 25ug/mL.
Yeast culture medium is YPD culture medium (1% yeast extract, 2% peptone, 2% glucose).Yeast screening assay culture
Base is YPD+Zeo culture medium (YPD+Zeo culture medium is the Zeocin that final concentration of 100ug/mL is added in YPD culture medium).
Yeast induced medium BMGY culture medium (1% yeast extract, 2% peptone, 1.34%YNB, 0.00004%
Biotin, 1% glycerol (V/V)) and BMMY culture medium (replacing glycerol divided by 0.5% methanol, remaining composition phase is identical as BMGY).
The basic salt culture medium of recombination yeast fermented and cultured: diammonium hydrogen phosphate 5%, potassium dihydrogen phosphate 0.5%, epsom salt
1.5%, potassium sulfate 1.95%, calcium sulfate 0.1%, potassium hydroxide 0.1%, defoaming agent 0.03%.1L adds 4.35ml after high pressure
PTM1。
PTM1 (Trace salts solution): copper sulphate 0.6%, potassium iodide 0.018%, manganese sulfate monohydrate 0.3%, Sodium Molybdate Dihydrate
0.02%, boric acid 0.002%, CoCL2 6H2O 0.05%, zinc chloride 2%, green-vitriol 6.5%, the concentrated sulfuric acid 0.5%, life
Object element 0.02%.
Do not make the experimental methods of molecular biology illustrated in following embodiment, referring to " Molecular Cloning:A Laboratory guide "
Listed specific method carries out in one book of (third edition) J. Pehanorm Brooker, or carries out according to kit and product description；
The reagent and biomaterial commercially obtain unless otherwise specified.
Embodiment 1, the synthesis of phytase gene and clone
Using the amino acid sequence of Kosakonia sacchari phytase KsPHY as with reference to (such as SEQ ID NO.1 institute
Show), artificial synthesized KsPHY gene.
According to gene order design primer, the end 5' introduces Nde I restriction enzyme site, and the end 3' introduces EcoR I restriction enzyme site, draws
Object sequence is as follows:
The end 5' primer Nde I-KsPHY-F1:
GGATTACCATATGAAAGAAGACTCCGCAATGAAGCTGG
The end 3' primer EcoR I-KsPHY-R1:CGGAATTCTCATAAGCCGCAATCAGCGACGCGC
Using the KsPHY gene of synthesis as template, PCR amplification is carried out, obtains the DNA band that size is about 1.2kb, recycles mesh
Segment, carry out double digestion with Nde I and EcoR I, be connected to linear pET-22b (+) carrier with identical restriction enzyme site
On, conversion to Top10 Escherichia coli, LB+Amp plate culture obtains pET-22b-KsPHY positive bacterium colony.
Embodiment 2, gene site-directed saturation mutation
Phytase 12 are determined to mutational site, is respectively as follows: the 73rd A, the 80th A, the 107th D, the 203rd
N, the 211st G, the 224th Q, 253rd Q, the 290th I, the 326th Y, 340th V of position, the 379th
K targetedly devises 12 pairs of saturation mutation primers, and target gene mutational site is unified for NNS, and NNS or so respectively takes 15 alkali
Base constitutes forward primer；Reverse primer and forward primer complete complementary, wherein N represents tetra- kinds of bases of A, T, C, G, and S represents C, G
Two kinds of bases.
Using recombinant vector pET-22b-KsPHY as template, forward and reverse primer in mutational site is added,Super fidelity 2 ×
Master Mix PCR polymerase carries out PCR amplification, product electroporated OrigamiB Escherichia coli after Dpn I digestion processing
Competent cell, in LB+Amp plate screening Positive mutants recombinant clone.186 monoclonals of each mutational site picking are inoculated into
96 hole deep-well plates.Each plate selects 3 unmutated clones as control.Every hole culture medium containing 500uL LB+Amp.37 DEG C of shaking tables
200rpm cultivates 5h, and after shifting 50uL bacterium solution to 96 new hole deep-well plates preservation of bacteria strain, addition contains IPTG in remaining bacterium solution
50uL LB+Amp culture medium, and make the final concentration of 0.5mM of the IPTG in every hole, 37 DEG C of shaking table 200rpm stay overnight inducing expression plant
Sour enzyme.Containing the enzyme solution for being incubated overnight inducing expression phytase after 80 DEG C of water-baths heat 5min, detect in crude enzyme liquid
Remain phytase activity.The preliminary heat-resistant activity detection of phytase is according to National Standard of the People's Republic of China " GB/T 18634-
2002 " it carries out.
According to phytase activity testing result, clone's group that phytase temperature tolerance is higher than control is picked as positive colony.
Selected from strain plate positive colony focus on a 96 hole deep-well plates repeat above-mentioned culture, inducing expression, enzyme activity determination sieve
Choosing test.It determines that temperature tolerance raising is Positive mutants clone, extracts positive colony plasmid DNA and carry out gene sequencing.
By fixed point saturation mutation after obtain 18 positive colonies, be respectively: A73F, A73V, A73P, T73D, A80P,
A80Y、D107K、D107P、D107A、N203D、G211S、Q224E、Q252V、Q253E、T326Y、K379P、K379A、K379Q。
For the phytic acid enzyme mutant for further obtaining high-temperature resistant, the present invention carries out the random groups of two o'clock or multi-point to these mutational sites
It closes, and has carried out the screening of the recombination between mutational site.Multipoint mutation carries out mutation gradually according to the method for single-point rite-directed mutagenesis,
Forward mutation assay is determined according to phytase heat-resistant activity testing result, in conjunction with high flux screening, obtains the phytic acid of a plant height heatproof
Enzyme mutant KsPHY9.
The building of embodiment 3, phytase KsPHY9 yeast expression vector
According to phytase KsPHY9 gene design primer, the end 5' introduces EcoR I restriction enzyme site, and the end 3' introduces Not I digestion
Site, primer sequence are as follows:
The end 5' primer EcoR I-KsPHY9-F1:GTAGAATTCAAAGAAGACTCCGCAATGAAGCTGG
The end 3' primer Not I-KsPHY9-R1:ATTGCGGCCGCTCATAAGCCGCAATCAGCGACGCGC
Using phytic acid enzyme mutant KsPHY9 gene as template, PCR amplification is carried out with above-mentioned primer, obtaining size is about
The DNA band of 1.2kb recycles target fragment, carries out double digestion with EcoR I and Not I, be connected to identical restriction enzyme site
Linear pPICZ α A carrier on, so that phytic acid enzyme mutant KsPHY9 gene is inserted into the signal peptide sequence of above-mentioned expression vector
Downstream.Connection product converts Top10 Escherichia coli, and LB+Zeo plate culture obtains pPICZ α A-PHd positive bacterium colony, extracts
PPICZ α A-KsPHY9 positive bacterium colony plasmid.After the linearisation of Pme I restriction enzyme, electroporated Pichia pastoris X33
Competent cell is coated with the YPDS solid culture plate of the Zeocin containing 100ug/mL, 30 DEG C of culture 2-3d.Picking as far as possible
The speed of growth is fast on Zeocin YPDS plate and the biggish transformant of bacterium colony, and 66 transformants of each plate picking are in 24 hole depths
Orifice plate, each deep-well plates contain 22 transformants, and the original transformation bacterium before 2 transformations is as control.Every hole yeast culture medium containing 2mL
BMGY grows to saturation state to it, and centrifugation discards BMGY culture medium, changes yeast induced medium BMMY, induces 24 hours
Afterwards, supernatant is taken to carry out phytase activity detection and heat-resistant activity detection.
The thermal stability of embodiment 4, original phytase and mutation phytase compares
The determination of activity of phytase is carried out according to National Standard of the People's Republic of China " GB/T 18634-2009 ".Phytic acid
Enzymatic activity definition refers to that sample is per minute in 5.0mmol/L sodium phytate from concentration under conditions of 37 DEG C of temperature, pH value 5.5
L μm of ol Phos is discharged, an as phytase activity unit is indicated with U.
U=FxC/ (Vx30)
In formula: the activity of phytase, U/mL in U- sample；C- is according to the light absorption value of practical sample liquid by linear regression equation meter
The enzymatic activity of calculating, U；Total extension rate before the reaction of F- sample solution；V- volume of sample, mL；30- reaction time, min.
The formulation of standard curve such as table 1:
| Phosphorus concentration (μm ol/L) | 0 | 1.5625 | 3.125 | 6.25 | 12.5 | 25 |
| OD value | 0 | 0.054 | 0.109 | 0.212 | 0.437 | 0.942 |
Phytase enzyme solution is placed in teat glass, is heat-treated 5min at 80 DEG C, is control with untreated enzyme activity,
Measurement residual phytase activity, enzyme activity after heat treatment by comparison to get to remaining enzyme activity at this temperature.
| Enzyme | 80 DEG C of water-bath 5min enzyme activity retention rates |
| KsPHY9 enzyme solution | 56.9% |
| Parent's KsPHY enzyme solution | 23.7% |
The optimal reaction pH of embodiment 5, original phytase and mutation phytase compares
Phytase optimal pH: extracting with different pH buffers and be diluted to certain enzyme activity for the phytase of known enzyme activity, and
It is measured with corresponding pH substrate according to National Standard Method and calculates opposite enzyme activity (%).Every group parallel to three.As shown in figure 3, original plant
Sour enzyme is consistent with the optimal pH of mutant.
The degrees of embodiment 6, original phytase and mutation phytase compare
The different pH buffers of the phytase of known enzyme activity are extracted and be diluted to 20U, are placed in 4 in 37 DEG C of 150rpm shaking tables
It is placed in mixture of ice and water at once after hour, according to National Standard Method dilution metering and calculates opposite enzyme activity (%).Every group flat to three
Row.As shown in figure 4, original phytase is consistent with the degrees of mutant.
Embodiment 7,7L fermentor lab scale
From the single colonie of picking engineering bacteria KsPHY9 on YPD+Zeo plate, it is inoculated in 20mL BMGY culture medium, 30 DEG C,
240rpm cultivates 20h.It is inoculated into 300mL BMGY culture medium with the ratio of 1:50,30 DEG C, 240rpm culture, to bacterium solution OD600
When about 0.5, for being seeded in fermentor.In fermentation process, temperature is controlled at 30 DEG C, and ventilatory capacity maintains 2vvm, revolving speed
Control is between 500-800rpm to maintain 20% or more dissolved oxygen.
Fermentation process is divided into three phases:
1) the strain culturing stage: the addition 3L fermentation basal medium in domestic 7L fermentor, 121 DEG C of sterilizing 20min,
30 DEG C are adjusted the temperature to, pH to 4.6 is adjusted with ammonium hydroxide, is added PTMl (4.35mL/L), access kind daughter bacteria (1:10), air agitation
About 18-24h is cultivated, until dissolved oxygen being shown as glycerol depletion in fermentor and being flown up；
2) it is long-term to enter glycerol growth-promoting, adds 50% glycerol (containing PTMl, 12mL/L), feed rate 18mL/Lh,
Continue 4-6h；
3) induction period is finally entered, with ammonium hydroxide or phosphorus acid for adjusting pH to desirable value, stream plus 100% methanol (containing PTMl,
12mL/L), flow velocity linearly rises to 4mL/Lh through 15hr from 1mL/Lh, continues 120h.
In fermentation process, every take for 24 hours fermentation liquid measure OD600And thallus weight in wet base, take supernatant to carry out phytase activity
Detection.The final average fermentation enzyme activity of fermentation ends reaches 19700U/mL.
Sequence table
<110>GuangDong YiDuoLi Biology Science Co., Ltd
<120>the phytic acid enzyme mutant KsPHY9 of high thermal stability and its gene and application
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 409
<212> PRT
<213> Kosakonia sacchari
<400> 1
Lys Glu Asp Ser Ala Met Lys Leu Glu Arg Val Val Ile Val Ser Arg
1 5 10 15
His Gly Val Arg Ala Pro Thr Lys Phe Thr Pro Gln Met Arg Glu Val
20 25 30
Thr Pro Phe Gln Trp Pro Gln Trp Glu Val Pro Leu Gly Trp Leu Thr
35 40 45
Pro Arg Gly Gly Gln Leu Ile Ser Ala Leu Gly His Tyr Gln Arg Leu
50 55 60
Arg Leu Ala Asp Ala Gly Leu Leu Thr Asp Lys Thr Cys Pro Asp Ala
65 70 75 80
Gly Arg Val Ala Val Ile Ala Asp Thr Asp Gln Arg Thr Arg Lys Thr
85 90 95
Gly Glu Ala Phe Leu Thr Gly Leu Ala Pro Asp Cys Pro Ile Pro Val
100 105 110
His Tyr Gln Gln Glu Lys Ser Lys Thr Asp Pro Leu Phe Asn Pro Ile
115 120 125
Lys Thr Gly Lys Cys Ala Phe Asn Thr Ser Ala Val Lys Glu Ala Ile
130 135 140
Leu Thr Lys Ala Gly Gly Asn Ile Glu Gln Tyr Thr Gln Lys Tyr Gln
145 150 155 160
Pro Ala Phe Gln Ala Leu Glu His Val Leu Asn Phe Pro Val Ser Glu
165 170 175
Lys Cys Lys Thr Ala Gly Glu Asn His Val Cys Ser Phe Thr Arg Asp
180 185 190
Ile Pro Thr Lys Leu Asn Ile Arg Pro Asp Asn Val Ser Leu Pro Gly
195 200 205
Ala Trp Gly Leu Ser Ser Thr Leu Thr Glu Ile Phe Leu Leu Gln Gln
210 215 220
Ala Gln Gly Met Thr Asp Val Ala Trp Gly Arg Ile Ala Gly Asp Lys
225 230 235 240
Glu Trp Gln Ser Leu Leu Ser Leu His Asn Ala Gln Phe Asp Leu Leu
245 250 255
Gln Arg Thr Pro Glu Val Ala Arg Ser Arg Ala Thr Pro Leu Leu Asp
260 265 270
Leu Ile Arg Thr Ala Leu Thr Thr Asn Gly Ala Asp Gln Asn His Tyr
275 280 285
Gly Ile Thr Phe Pro Val Ser Val Leu Phe Ile Ala Gly His Asp Thr
290 295 300
Asn Leu Ala Asn Leu Ser Gly Ala Leu Asp Leu Asn Trp Ser Leu Pro
305 310 315 320
Ser Gln Pro Asp Asn Thr Pro Pro Gly Gly Glu Leu Val Phe Glu Arg
325 330 335
Trp Lys Arg Val Ser Asp Asn Thr Asp Trp Val Gln Val Ser Phe Val
340 345 350
Tyr Gln Thr Leu Gln Glu Met Arg Glu Met Arg Ala Phe Ser Arg Asp
355 360 365
Asn Pro Pro Gly Arg Val Asp Leu Ala Leu Lys Ala Cys Ser Glu Lys
370 375 380
Asn Ala Gln Gly Met Cys Ser Leu Ser Ser Phe Ala Lys Leu Ile Glu
385 390 395 400
Ser Leu Arg Val Ala Asp Cys Gly Leu
405
<210> 2
<211> 409
<212> PRT
<213>artificial sequence (Artificial Sequence)
<400> 2
Lys Glu Asp Ser Ala Met Lys Leu Glu Arg Val Val Ile Val Ser Arg
1 5 10 15
His Gly Val Arg Ala Pro Thr Lys Phe Thr Pro Gln Met Arg Glu Val
20 25 30
Thr Pro Phe Gln Trp Pro Gln Trp Glu Val Pro Leu Gly Trp Leu Thr
35 40 45
Pro Arg Gly Gly Gln Leu Val Ser Ala Leu Gly His Tyr Gln Arg Leu
50 55 60
Arg Leu Ala Asp Ala Gly Leu Leu Thr Asp Lys Thr Cys Pro Asp Pro
65 70 75 80
Gly Arg Val Ala Val Ile Ala Asp Thr Asp Gln Arg Thr Arg Lys Thr
85 90 95
Gly Glu Ala Phe Leu Ala Gly Leu Ala Pro Asp Cys Pro Ile Pro Val
100 105 110
His Tyr Gln Gln Glu Lys Ser Lys Thr Asp Pro Leu Phe Asn Pro Ile
115 120 125
Lys Thr Gly Lys Cys Ala Phe Asn Thr Ser Ala Val Lys Glu Ala Ile
130 135 140
Leu Thr Lys Ala Gly Gly Asn Ile Glu Gln Tyr Thr Gln Lys Tyr Gln
145 150 155 160
Pro Ala Phe Gln Ala Leu Glu His Val Leu Asn Phe Pro Val Ser Glu
165 170 175
Lys Cys Lys Thr Ala Gly Glu Asn His Val Cys Ser Phe Thr Arg Asp
180 185 190
Ile Pro Thr Lys Leu Asn Ile Arg Pro Asp Asp Val Ser Leu Pro Gly
195 200 205
Ala Trp Ser Leu Ser Ser Thr Leu Thr Glu Ile Phe Leu Leu Gln Glu
210 215 220
Ala Gln Gly Met Thr Asp Val Ala Trp Gly Arg Ile Ala Gly Asp Lys
225 230 235 240
Glu Trp Gln Ser Leu Leu Ser Leu His Asn Ala Val Phe Asp Leu Leu
245 250 255
Gln Arg Thr Pro Glu Val Ala Arg Ser Arg Ala Thr Pro Leu Leu Asp
260 265 270
Leu Ile Arg Thr Ala Leu Thr Thr Asn Gly Ala Asp Gln Asn His Tyr
275 280 285
Gly Ile Thr Phe Pro Val Ser Val Leu Phe Ile Ala Gly His Asp Thr
290 295 300
Asn Leu Ala Asn Leu Ser Gly Ala Leu Asp Leu Asn Trp Ser Leu Pro
305 310 315 320
Ser Gln Pro Asp Asn Tyr Pro Pro Gly Gly Glu Leu Val Phe Glu Arg
325 330 335
Trp Lys Arg Val Ser Asp Asn Thr Asp Trp Val Gln Val Ser Phe Val
340 345 350
Tyr Gln Thr Leu Gln Glu Met Arg Glu Met Arg Ala Phe Ser Arg Asp
355 360 365
Asn Pro Pro Gly Arg Val Asp Leu Ala Leu Pro Ala Cys Ser Glu Lys
370 375 380
Asn Ala Gln Gly Met Cys Ser Leu Ser Ser Phe Ala Lys Leu Ile Glu
385 390 395 400
Ser Leu Arg Val Ala Asp Cys Gly Leu
405
Claims (7)
1. the phytic acid enzyme mutant KsPHY9 that thermal stability improves, which is characterized in that the ammonia of the phytic acid enzyme mutant KsPHY9
Base acid sequence is that amino acid sequence phytase KsPHY as shown in SEQ ID NO:1 includes following mutational site: A73P, A80P,
D107K, N203D, G211S, Q224E, Q252V, T326Y, K379P.
2. the phytic acid enzyme mutant gene that thermal stability improves, which is characterized in that the gene encodes heat described in claim 1
Stability-enhanced phytic acid enzyme mutant KsPHY9.
3. the recombinant vector comprising the phytic acid enzyme mutant gene that thermal stability as claimed in claim 2 improves.
Expression vector for expressing the phytic acid enzyme mutant is pPICZ α A and pPIC9K；Place for the expression vector
Chief cell is.
4. the recombinant vector pPICZ α A- containing the phytic acid enzyme mutant gene that thermal stability as claimed in claim 2 improves
KsPHY9。
5. the recombinant bacterial strain of the recombinant vector of the phytic acid enzyme mutant gene containing thermal stability raising as claimed in claim 2.
6. the recombinant bacterial strain containing the phytic acid enzyme mutant gene that thermal stability as claimed in claim 2 improves, which is characterized in that institute
Stating bacterial strain is Pichia pastoris X33 or GS115.
7. a kind of method for the phytic acid enzyme mutant KsPHY9 for preparing the raising of thermal stability described in claim 1, which is characterized in that
It is described the following steps are included:
1) host cell is converted with recombinant vector as claimed in claim 3, obtains recombinant bacterial strain；
2) it ferments to recombinant bacterial strain, induces the expression of recombinant phytase.
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